M465:Gel electrophoresis and western blots

Agarose Gel Electrophoresis of Your wsp PCR product

To see if you successfully amplified the wsp gene - and therefore identified Wolbachia in your samples - you will "run a gel" on your pcr products. To run a gel means that we will perform an electrophoretic separation of the DNA fragments in your pcr product, using a small fraction of the volume of your pcr product applied to a 1% agarose gel stained with Sybr Safe DNA stain. Your instructor will help you to photograph the gel so you can evaluate whether you found the Wild Wolbachia. You should see a single band of ~600 bp if Wolbachia was present.

Quick Q: Can you explain how we know the size of our amplified gene fragment?

Protocol for making a 1% agarose gel

In order to run out your PCR products you will first pour a 1% agarose gel. The recipe for the gel is 1.0% agarose (w/v) in 1x TBE buffer (10x=890mM Tris, 890mM Boric Acid, 20mM EDTA) with SybrSafe™ stain. Your instructor will add the SybrSafe stain to your molten agarose after you prepare it. Before you start, prepare your agarose gel, comb, and reservoir as you are instructed. You will share one gel per four students - this will provide you with the number of lanes adequate to accommodate all your PCR reactions.

Protocol: 1. Using a graduated cylinder, fill a glass, 250 mL flask with 100 mL of 1x TBE buffer.
2. Using the top loading balance, measure out 1 gram of Agarose
3. Pour the agarose into the flask containing the TBE buffer and stir to mix. The agarose won't go into solution here but needs to be heated
4. Place the flask inside the microwave and heat for 1 minute, maximum power. At the end of the minute, swirl the solution to mix and place back in the microwave.
5. Heat for 1 minute again, this time watching closely to make sure it does not boil. At the end, swirl to mix and bring to your instructor for the sybersafe dye
6. After your instructor adds the SyberSafe, pour your gel into the mould. It will take ~ 25 minutes to set.

DNA is uniformly negatively charged and will,therefore, move toward the positive electrode. The separation is determined by the size or mass of the molecule or fragments of DNA.

Procedure for Agarose Gel Electrophoresis of PCR products
1. Load 5 microliters of your PCR product on your gel by placing the 5 microliters of your pcr product as a spot on a small piece of parafilm and adding 5 microliters of loading dye (0.25% XC, 30% glycerol, 0.1mg/ml RNAase).
2. Mix the loading dye by pipetting up and down on the spot, on the parafilm, before loading all 10 microliters into a lane of the 1% agarose gel.
3. Record which lane is loaded with which product. Be sure to leave the first two lanes and the last lane empty for the 100 bp ladder. Do not forget to load your positive and negative controls as well!
4. The gel will be run at 120V for approximately 30 minutes.

Western Blots